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Beverly Wendland

James B. Knapp Dean of the Krieger School of Arts & SciencesProfessor

In May 2014, Provost Robert Lieberman appointed Beverly Wendland, PhD, as interim dean of the Krieger School of Arts and Sciences. On February 12, 2015, Johns Hopkins University President Ronald J. Daniels announced that the executive committee of the board of trustees had approved his recommendation that Dr. Wendland be appointed the new James B. Knapp Dean of the Krieger School.

A Johns Hopkins faculty member since 1998, Dr. Wendland has a strong knowledge of the Krieger School and its operations. She became chair of the Department of Biology in 2009, leading faculty, staff, and students during a period of renewal. She also holds a joint appointment in the Department of Biophysics.

Dr. Wendland earned her bachelor’s degree in bioengineering at the University of California, San Diego, and her doctoral degree in neurosciences from Stanford University. She joined the Department of Biology at Johns Hopkins after completing her postdoctoral studies at the University of California, San Diego.

A recipient of funding from the National Institutes of Health and the National Science Foundation, Dr. Wendland and her team study fundamental cellular processes using yeast cells as a simple model system. Discoveries about how yeast cell function can also teach us about human diseases, such as neurodegenerative diseases or cancer. Her lab’s work may ultimately identify new targets for treatments, such as enhanced delivery of gene therapies.

Throughout her tenure at Johns Hopkins, Dr. Wendland has supported graduate and undergraduate students, serving as their mentor and a collaborator. An advocate for training future scientists to engage the power of interdisciplinary research, she was a member of the lab advisory committee during the construction of the new Undergraduate Teaching Laboratories, designed to foster collaboration across Homewood’s scientific community. Dr. Wendland was also a member of the Krieger School’s advisory committee on the status of women.

Molecular Mechanisms and Regulation of Endocytic Vesicle Formation

Endocytosis regulates cell physiology and homeostasis by affecting nutrient uptake, signal transduction, and the population of receptors in the plasma membrane. Cellular outputs are in turn affected, including cell fate decisions, cell division/proliferation, and cell polarity. Understanding the mechanisms of endocytosis will therefore lead to a better understanding of its function in these fundamental cellular processes and reveal how endocytosis interfaces with other processes to affect the response of a cell to its environment. Because many components of the endocytic machinery are structurally and functionally conserved from yeast to humans, we are able to apply the many powerful tools available in the yeast system to develop new experimental approaches and generate more accurate models to decipher the endocytic pathway. We anticipate that our discoveries in yeast will inform studies of human diseases that arise from aberrant endocytic regulation, including some forms of cancer, and provide new targets for therapeutics, such as enhanced delivery of gene therapies.

We focus our studies on clathrin-dependent endocytosis, which can be divided into three stages. A) A specific subset of membrane proteins (cargo) is selected for incorporation into the endocytic vesicle. Endocytic proteins called “adaptors” regulate this cargo recruitment through binding to sorting motifs in the cytoplasmic domains of the cargo. B) The structural components of the endocytic vesicle are recruited, including the “coat” protein clathrin and the “scaffold” proteins that coordinate the proper association of adaptors and coats. We have recently shown that scaffolds link the early events of vesicle formation to the late events of vesicle scission, a new role for these proteins. C) The formed endocytic vesicle then separates from the plasma membrane (scission) and moves into the cytosol. Completion of both vesicle scission and movement require the actin cytoskeleton.

Our long-term goal is to understand the molecular mechanisms that drive each step of endocytosis and to determine how these individual steps are coordinated to occur with the proper order and timing. Toward this goal, our lab has focused on two broad areas: 1) Defining which proteins act as adaptors, how they regulate cargo selection, and if they have other functions in endocytosis. 2) Determining the role and mode of action of the endocytic scaffold Pan1 in coordinating the sequential events in endocytosis.us (LIDL-COO-) mimics the canonical ‘clathrin box’ (LLDLD) of mammalian adaptors that binds the terminal domain of clathrin. In addition to binding clathrin, we and others showed that the epsins contain multiple motifs with distinct binding activities, including the lipid-binding ENTH domain, UIMs that bind ubiquitin, and NPF motifs that bind the EH domains of the scaffolds Pan1 and Ede1. Using a novel in vitro membrane recruitment assay, we obtained evidence that supports the view that the ENTH domain and UIM motifs act together through their individual low affinity interactions to create a stable multivalent complex at endocytic sites. These and other findings in the field led to the model that epsins can act as endocytic adaptors that play a key role in initiating endocytic events.

Since adaptors likely play a major role in controlling cell physiology, two key questions are how are adaptors regulated, and do they have additional functions? Since phosphorylation is known to regulate the mammalian endocytic machinery, we asked if/how phosphorylation regulates yeast endocytic adaptors. We determined that epsins are phosphorylated in vivo by the conserved protein kinase Prk1, and that this modification inhibits epsin functions. We also found that the epsins and the structurally related Yap180 proteins fulfill a redundant function in endocytosis and that this function requires their NPF motifs, which bind EH domains in scaffold proteins. Our exciting findings suggest a new function for adaptors, that were thought to be primarily early-acting factors, in regulating the onset of the final stages of vesicle scission (see below).

We next asked if the essential function of the epsins was connected to their role in support of endocytosis, or to some other process. We and others have shown that the conserved N-terminal 'ENTH' domains of the epsins bind phosphoinositides, while the epsin C-termini harbor the motifs for binding to other endocytic machinery components. Thus, we were surprised to find that the ENTH domain alone was necessary and sufficient to complement both endocytosis defects and inviability of ent1∆ent2∆ cells. A yeast two-hybrid screen with the ENTH domain led to our discovery of a novel essential role for ENTH domains that is independent of lipid-binding: down-regulation of the GTPase Cdc42 by binding to Cdc42 GAPs. Another recent screen in our lab has identified a genetic interaction between the adaptors and the GTPase Rho1. The Rho-family GTPases Cdc42 and Rho1 are critical regulators of cell polarity and the actin cytoskeleton. These findings connecting endocytic adaptor proteins and Cdc42/Rho1 may explain the correlation of sites of polarity cues, secretion, and endocytosis. Our future studies will define the mechanisms by which signaling pathways activated by these GTPases may regulate endocytosis, how the adaptors may serve as a critical interface, and if these interactions directly or indirectly impact endocytosis.

Endocytic scaffold protein functions: Our discovery of the scaffold protein Pan1 as a critical endocytic factor in yeast revealed the conserved, universal aspects of eukaryotic endocytic mechanisms. Subsequent data from our lab and others have led us to hypothesize that Pan1 is a central regulator or checkpoint protein that controls the transitions between early and late stages of endocytosis. Thus, understanding the molecular mechanisms of Pan1 function continues to be a major focus of our work. Specifically, we are testing the hypothesis that a Pan1:adaptor:cargo complex can sense the completion of cargo-loading, consequently triggering the recruitment and activation of the actin-based scission machinery. Consistent with this model, evidence from our lab and others shows that Pan1 interacts with factors important for both early stages (cargo collection) and late stages (vesicle scission) of endocytosis. Like Pan1, the EH domain-containing scaffold/adaptor protein Ede1 also binds the epsin and Yap180 adaptors; thus, we are studying the unique and shared functions of Pan1 vs. Ede1. We also demonstrated that Pan1 binds directly to the late-acting type I myosins (Myo3/5), and that Pan1 stimulates the actin-assembly activity of Myo5/Vrp1 at the time when vesicle invagination/scission commences. Additionally, we showed that Pan1 forms homo-oligomers and exhibits intra-molecular interactions between distinct domains that may also regulate Pan1 functions. Our ongoing and future studies are centered on clarifying the exchange of binding partners during the sequential formation and dissolution of Pan1-containing protein complexes, using biochemical, biophysical and cell biological approaches. In this way we will test our model for how Pan1 may act as a checkpoint protein to control the progression from one endocytic stage to the next.

Matching cargos to adaptors: In our work on endocytic adaptors, we are identifying known and novel proteins that can act as adaptors and studying the mechanism(s) by which they fulfill their important functions. Adaptors have several characteristics, including binding directly to sorting signals in the tail of the transmembrane cargo that is to be endocytosed, promoting clathrin polymerization, and more. The wide variety of transmembrane cargo proteins suggests that a range of adaptors mediate cargo endocytosis, yet many of these adaptors remain unknown. Thus, we are trying to identify endocytic plasma membrane cargos and the sorting signals recognized by their cognate adaptors. For example, we have evidence that Yap1801 and Yap1802 (AP180/CALM homologs; recently implicated in Alzheimer’s Disease) are cargo-specific adaptors for the v-SNAREs Snc1/2 (VAMP/synaptobrevin homologs). Other candidate adaptors we are studying include the AP-2 complex, the ENTH domain protein Ent4, and the novel conserved protein Syp1. We have recently begun to apply structural biology tools to our questions, and found that Syp1 not only has a domain common to adaptor proteins, but it also has a homo-dimeric membrane tubulation domain. Additionally, while assessing the requirements and roles for adaptors in vivo using a genetic screen, we uncovered an allele of the gene encoding Sla2. Sla2 is critical for endocytosis (Riezman lab) and binds clathrin (Lemmon lab); these two results together with our findings are consistent with the model that Sla2 may be another endocytic adaptor.

Developing new tools to study endocytosis: To better define the functions and regulation of endocytic adaptors and scaffolds, we are applying tools used in studies of synaptic vesicle recycling to studies of yeast endocytosis. For example, to develop a cargo-specific, quantitative, high-throughput method to screen for endocytosis defects, we are using pH-sensitive GFP variants fused to endocytic cargo. This allows for quenching of the vacuolar GFP fluorescence derived from endocytosed receptors, so that the signal from uninternalized receptors can be selectively observed.

The combination of genetic, biochemical, cell biological and biophysical approaches used by our lab will allow us to uncover new factors and mechanism of endocytosis, and to test our model for Pan1 regulation of endocytosis by defining the sequential conformations and complexes that underlie Pan1’s functions. Our current and future directions will add clarity to mechanistic models of endocytic protein function. Overall, our lab’s work has contributed to elucidating the fundamental mechanisms of endocytosis in all eukaryotic cells. Our ongoing and future work will continue to shed light on the conserved functions and regulation of endocytosis and its role in cellular physiology, homeostasis and pathological conditions, including cancer, cardiovascular disease, lysosomal storage disorders, and infections by viral and bacterial pathogens.